Project Summary (2000-2005)

A protein analysis and imaging core shall be established to assist the investigators in characterizing novel protein; assaying bound metals in protein-metal complexes; and assaying phosphorylation states and alkylphosphoryl-conjugates of proteins. The Core shall be administered by Elizabeth Komives, who will implement new methods for mass spectrometric characterization of proteins, and Mark Ellisman, who will employ confocal and electron microscopy, coupled with recombinant DNA and immunohistochemistry techniques, to provide high resolution analysis of tissues from transgenic animals prior to following toxicant exposure. The specific aims of the Core include:

1. Identification of novel proteins isolated from 20 gels, immunoprecipitates and glutathione pull-down experiments. MALDI mass spectrometry will be used to provide tryptic maps and databases will be searched for proteins with similar maps.
2. Protein phosphorylation states will be analyzed as intact proteins to determine sequential phosphorylation. Also tryptic and endo-C digestions will be used to localize phosphorylation sites by mass spectrometry. A similar approach will be used for the alkylphosphoryl conjugates where MAALDI will be used to quantitate the phosphorylated and unphosphorylated protein in immunoprecipitates, while nanospray on the peptides will be used when abundance is low (QSTAR with Protana nanospray).
3. Protein-metal complex involved in heavy metal detoxification shall be characterized in an electrospray ion and rap mass spectrometer. Mass spectrometry will allow metal identification and determination of stoichiometry. The low voltages of the electrospray allow the metal complexes to survive the mass spectrometer source.
4. Oxidation slides of cysteines in proteins, as an oxidative stress response, will be quantitated by initial alkylation of the reactive cysteines and mass spectral analysis of the alkylation products.
5. The imagine core will provide assistance in confocal microscopy, immunofluorescence, in situ hybridization, EM immunolocalizations and tomography and multidimensional image analysis interfaced with cryoelectronmicroscopy. These approaches will follow the initial histopathology.